Abstract

Jasmonates are a family of plant hormones that regulate plant growth, development and responses to stress. The F-box protein CORONATINE INSENSITIVE 1 (COI1) mediates jasmonate signalling by promoting hormone-dependent ubiquitylation and degradation of transcriptional repressor JAZ proteins. Despite its importance, the mechanism of jasmonate perception remains unclear. Here we present structural and pharmacological data to show that the true Arabidopsis jasmonate receptor is a complex of both COI1 and JAZ. COI1 contains an open pocket that recognizes the bioactive hormone (3R,7S)-jasmonoyl-l-isoleucine (JA-Ile) with high specificity. High-affinity hormone binding requires a bipartite JAZ degron sequence consisting of a conserved α-helix for COI1 docking and a loop region to trap the hormone in its binding pocket. In addition, we identify a third critical component of the jasmonate co-receptor complex, inositol pentakisphosphate, which interacts with both COI1 and JAZ adjacent to the ligand. Our results unravel the mechanism of jasmonate perception and highlight the ability of F-box proteins to evolve as multi-component signalling hubs.

a. Binding of tritium-labeled coronatine (300 nM) to recombinant COI1-ASK1 and JAZ proteins. b. Saturation binding of 3H-coronatine to the complex of COI1-ASK1 in the presence of JAZ6 or JAZ1, with a Kd of 68±15 nM and 48±13 nM, respectively. c. Chemical structures of (3R,7S)-JA-Ile and coronatine. d. The consensus sequence of the Jas motif from 61 JAZ proteins from two monocot and three dicot plant species. Corresponding peptide sequences from JAZ1 in (e) are listed below. e. 3H-coronatine binding at 300 nM to COI1 in the presence of a series of synthetic JAZ1 peptides with the N-terminus of R205-Y226 systematically extended as described in (d). f. Saturation binding of COI1-ASK1 and the JAZ1 +5 degron peptide, with a Kd of 108±29 nM. All results are the mean ± S.E. of two to three experiments performed in duplicate.

Crystal structure of the COI1-ASK1 complex with JA-Ile and the JAZ degron peptide

a, b. COI1-ASK1 (green and grey ribbon, respectively), with JAZ degron peptide (orange ribbon) and (3R,7S)-JA-Ile in yellow spacefill. c. Surface representation of COI1 (grey) with Loops-2 (blue), -12 (purple) and -14 (green) forming the JA-Ile binding pocket. d, e. Side view of (3R,7S)-JA-Ile (JA-Ile) and coronatine (COR) binding. Hormones are shown as stick models, along with positive Fo-Fc electron density, calculated before they were built into the model (red mesh). Hydrogen bond and salt bridge networks are shown with yellow dashes. f. Top view of JA-Ile pocket showing the Fo-Fc electron density, calculated before JA-Ile was built into the model (red mesh). The electron density of the pentenyl side chain of (3R,7S)-JA-Ile cannot accommodate the (3R,7R)-JA-Ile side chain, which is constrained by the chiral configuration at the C7 position. g. When bound to COI1, JA-Ile (yellow spacefill) is solvent accessible at both the keto group (top) and carboxyl group (bottom).

a. Top view of the complete JAZ1 degron peptide (orange) bound to COI1 (green) and JA-Ile (yellow). b. Side view and surface representation of the JAZ peptide, which acts as a clamp to lock JA-Ile in the pocket. c. Interactions of the N-terminal region of the JAZ1 degron with COI1 and JA-Ile. Hydrogen bonds are shown with yellow dashes. d. Structural role of the Arg206 residue from the JAZ1 degron in coordinating the carboxyl group of JA-Ile with three basic residues of the COI1 ligand pocket floor. e. Top view of the amphiphathic JAZ1 degron helix bound to COI1 with three hydrophobic residues of JAZ1 shown in orange stick, and COI1 residues in colored surface representation. f. Coronatine-induced interactions of wild type and mutant COI1 with JAZ1 detected by yeast two-hybrid assay (sdm: site-directed mutants). Blue color indicates interaction.

a. Nano-electrospray MS of the intact COI1-ASK1 complex. Low intensity charge series corresponds in mass to the cofactor-free COI1-ASK1 complex. High intensity charge series corresponds to cofactor-bound COI1-ASK1 complex. b. Optimized cofactor purification scheme. c. Proton TOCSY spectrum of the purified cofactor. Numbers along the diagonal indicate the positions of the six protons of Ins(1,2,4,5,6)P5. The cross-peaks corresponding to direct couplings are labeled. Other cross-peaks correspond to relayed connectivities. d. TOCSY spectrum of a synthetic Ins(1,2,4,5,6)P5 as a standard. e. Islands of positive Fo-Fc electron density (red mesh) below the hormone-binding pockets, which likely belong to inorganic phosphate molecules from the crystallization solutions that displace InsP5 from the InsP5-binding site. f. Bottom view of a surface electrostatic potential representation of COI1 from positive (blue) to negative (red).

Inositol phosphate is an essential component of the COI1-JAZ co-receptor

a. Binding of 3H-coronatine at 100 nM to a complex of COI1 and JAZ1, with addition of 1 µM synthetic Ins(1,2,4,5,6)P5 (InsP5). b. With extensive dialysis to remove the co-purified InsP5 cofactor, 100 nM 3H-coronatine no longer binds dialyzed COI1 in the presence of JAZ1. Synthetic Ins(1,2,4,5,6)P5 rescues binding. c. Ins(1,2,4,5,6)P5 rescues the binding of 100 nM 3H-coronatine to dialyzed COI1-ASK1 in the presence of JAZ1 with an EC50 of 27±12 nM. d. Binding assays performed with 100 nM 3H-coronatine, dialyzed COI1, and 1 µM synthetic Ins(1,2,4,5,6)P5 (InsP5), Ins(1,4,5,6)P4 (InsP4), or Ins(1,4,5)P3 (InsP3). e. Saturation binding of 3H-coronatine to dialyzed COI1 in the presence of 1 µM of Ins(1,2,4,5,6)P5 (InsP5) and Ins(1,2,3,4,5,6)P6 (InsP6) at a Kd of 30±5 nM and 37±8 nM, respectively. All results are the mean ± S.E. of up to three experiments performed in duplicate. f. A phosphate-binding site in the complex structure reveals an interwoven hydrogen bond network that may explain the mechanism by which the InsP cofactor potentiates the JA co-receptor.